PROJECT SUMMARY The Fanconi Anemia (FA) pathway functions to maintain genomic stability and is crucial for the repair of DNA interstrand crosslinks (ICLs). During ICL repair, the central FANCD2 protein promotes the recruitment of downstream factors such as BRCA2[FANCD1] and RAD51[FANCR] that are crucial for homologous recombination (HR) repair of DNA double stranded breaks (DSBs), including those generated during ICL removal. Previous studies from our laboratory and others showed that FANCD2 has additional roles in promoting the HR-dependent recovery of hydroxyurea (HU) or aphidicolin (APH) stalled replication forks. Intriguingly, we recently identified a new constitutive FANCD2 interactor named ATRX (Alpha Thalassemia Retardation Syndrome X-linked). ATRX, in complex with DAXX (Death Domain-Associated Protein 6), acts as a chromatin remodeler and histone H3.3 chaperone that regulates chromatin compaction. While ATRX was originally not considered to be involved in mechanisms of genome maintenance, recent studies showed that acquired ATRX gene defects are strongly associated with a subset of cancers that exhibit excessive genome instability. However, the underlying mechanisms of ATRX-dependent genome stability are not known. To elucidate a potential functional cross-talk between the ATRX and FANCD2 pathways, we generated isogenic human knockout cell lines lacking ATRX, FANCD2, or both. We found that ATRX and FANCD2 form a constitutive protein complex with the MRE11-RAD50-NBS1 (MRN) nuclease. This complex acts as a functional unit to promote HR-dependent replication fork recovery and the repair of directly inducible DNA DSBs. Simultaneously, and paradoxically, we also observed that ATRX and FANCD2 gene defects had a synergistic effect on cellular ICL sensitivities. Based on our observations, we hypothesize that ATRX cooperates with FANCD2 and MRN to promote HR-mediated repair mechanisms, but also possesses additional activities that contribute to DNA ICL removal in the absence of a functional FA pathway. To test our hypothesis, we propose three Specific Aims: 1) Elucidate the molecular and structural makeup of the ATRX-MRN-FANCD2 protein complex 2) Determine molecular mechanisms of ATRX/FANCD2-mediated replication fork recovery. 3) Dissect FANCD2-dependent and -independent roles of ATRX during DNA ICL repair.